EXO70B2 Antibody

What is EXO70B2 and why is it relevant to immune response research?

EXO70B2 is a subunit of the exocyst complex that regulates the final steps of exocytosis and plays a crucial role in plant immunity. It functions as a molecular hub coupling the secretory machinery with immune signaling and autophagy. EXO70B2 has been shown to interact with and be phosphorylated by MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3), which affects its localization and function . This phosphorylation regulates EXO70B2's interaction with the plasma membrane and couples the secretory pathway with cellular signaling . Research has demonstrated that EXO70B2 is required for full activation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) . When studying plant immune responses, EXO70B2 antibodies can help track protein localization, abundance, and post-translational modifications that occur during defense activation.

What epitopes should be targeted when selecting or developing an EXO70B2 antibody?

When selecting epitopes for EXO70B2 antibody development, researchers should consider:

• The N-terminal domain, which shows less conservation among EXO70 family members and provides better specificity.

• Avoiding the C-terminal domain if studying truncation variants, as studies have shown that C-terminal truncation results in resistance to certain inhibitors like Endosidin2 (ES2) .

• Considering phosphorylation sites, particularly those targeted by MPK3, if studying post-translational modifications .

• Avoiding regions that may be masked during protein-protein interactions, especially ATG8-interacting motifs (AIMs) that mediate interaction with autophagy machinery .

Since the C-terminal domain may have distinct regulatory roles , antibodies targeting different regions of EXO70B2 might provide complementary insights into protein function and regulation.

How can I validate the specificity of an EXO70B2 antibody?

Comprehensive validation of EXO70B2 antibodies should include:

• Western blot analysis using multiple controls:

  • Wild-type samples versus exo70B2 mutant lines (like exo70B2-3)

  • Recombinant EXO70B2 protein as a positive control

  • Tissue samples from species expressing varying levels of EXO70B2

• Cross-reactivity assessment:

  • Testing against closely related proteins, particularly EXO70B1, which is the closest homologue and shares functional overlap

  • Evaluating reactivity against other EXO70 family members

• Immunoprecipitation validation:

  • Performing IP-MS (immunoprecipitation followed by mass spectrometry) to confirm pulldown of genuine EXO70B2 and known interactors like MPK3

  • Confirming the absence of nonspecific binding to other cellular components

• Immunofluorescence controls:

  • Comparing antibody staining patterns with GFP-tagged EXO70B2 localization

  • Peptide competition assays to demonstrate binding specificity

What techniques can be used to detect and quantify EXO70B2 expression levels?

Several techniques can be employed to detect and quantify EXO70B2:

• Immunoblotting (Western blot):

  • Most commonly used for detecting total protein levels

  • Can distinguish between different phosphorylation states when combined with Phos-tag gels

  • Useful for monitoring changes after treatments such as benzothiadiazole (BTH) or flg22

• Immunoprecipitation:

  • Essential for studying protein-protein interactions

  • Can be used to isolate EXO70B2 complexes for further analysis

  • Has been successfully used to demonstrate interaction with MPK3

• Immunofluorescence microscopy:

  • Allows visualization of subcellular localization

  • Particularly valuable for monitoring redistribution during immune responses

  • Can confirm plasma membrane association and vacuolar transport

• Fractionation combined with immunoblotting:

  • Enables assessment of EXO70B2 distribution between cellular compartments

  • Particularly useful for monitoring shifts between cytosolic, membrane-associated, and vacuolar pools

How should samples be prepared for optimal detection of EXO70B2?

Optimal sample preparation depends on the experimental approach:

• For total protein extraction:

  • Use buffer containing adequate detergent (0.5-1% Triton X-100) to solubilize membrane-associated EXO70B2

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation states

  • Maintain cold temperatures throughout extraction to minimize degradation

• For subcellular fractionation:

  • Sequential centrifugation can separate crude organellar fraction, soluble proteins, and microsomal fraction

  • This approach is particularly valuable when studying BTH-induced redistribution of EXO70B2

  • Include Concanamycin A (ConcA) treatment to inhibit vacuolar degradation when assessing vacuolar transport

• For immunolocalization:

  • Mild fixation (2-4% paraformaldehyde) preserves epitope accessibility

  • Consider using mannitol-induced plasmolysis to better visualize plasma membrane association

  • For root hairs, where EXO70B2 shows polar localization, capture images at different developmental stages

What controls should be included when using EXO70B2 antibodies in experimental workflows?

Proper experimental controls are essential for generating reliable data:

When studying immune responses, it's particularly important to include proper timing controls, as EXO70B2 dynamics change rapidly following treatment with immune elicitors .

How can I use EXO70B2 antibodies to study its phosphorylation state?

Studying EXO70B2 phosphorylation requires specialized approaches:

• Phospho-specific antibodies:

  • Consider developing antibodies that specifically recognize phosphorylated forms of EXO70B2, particularly at MPK3 target sites

  • These allow direct detection of phosphorylation state changes during immune responses

• Phos-tag SDS-PAGE:

  • This technique retards the migration of phosphorylated proteins

  • Can separate and quantify different phosphorylation states of EXO70B2

  • Compare migration patterns before and after phosphatase treatment to confirm phosphorylation

• Immunoprecipitation followed by phospho-detection:

  • Use anti-EXO70B2 antibodies to immunoprecipitate the protein

  • Probe with anti-phosphoserine/threonine antibodies

  • Alternatively, use phospho-proteomic mass spectrometry to identify specific phosphorylation sites

• Comparison with phospho-mimetic and phospho-null variants:

  • Generate transgenic lines expressing modified EXO70B2 (e.g., S-to-A or S-to-D mutations)

  • Compare antibody reactivity and localization patterns with these variants

  • This approach has revealed that phosphorylation affects both localization and autophagy targeting

What are the best methods for immunoprecipitation of EXO70B2 to study protein-protein interactions?

Effective immunoprecipitation of EXO70B2 requires careful consideration of:

• Buffer composition:

  • Use non-denaturing buffers to preserve protein-protein interactions

  • Include appropriate detergents (0.5-1% NP-40 or Triton X-100) to solubilize membrane-associated EXO70B2

  • Add protease and phosphatase inhibitors to prevent degradation and dephosphorylation

• Crosslinking strategies:

  • Consider mild crosslinking (0.5-1% formaldehyde) to stabilize transient interactions

  • This is particularly valuable for capturing interactions with components of the autophagy machinery

• Experimental design:

  • Include treatment conditions that modulate EXO70B2 interactions (e.g., BTH, flg22)

  • Compare wild-type and phospho-variant forms to assess the impact of phosphorylation on interactions

• Co-immunoprecipitation validation:

  • Confirm interactions by reciprocal co-immunoprecipitation

  • This approach has successfully demonstrated the interaction between EXO70B2 and MPK3

  • Consider using tagged versions (GFP-EXO70B2) as complementary approaches

How can I track changes in EXO70B2 localization during immune responses?

Monitoring EXO70B2 dynamics during immune responses requires:

• Time-course immunolocalization:

  • Perform immunostaining at multiple timepoints after immune stimulation

  • Include both short-term (minutes to hours) and long-term (hours to days) timepoints

  • Compare patterns after treatment with different immune elicitors (BTH, flg22)

• Co-localization with cellular markers:

  • Include markers for target compartments (PM, endosomes, vacuole)

  • Co-stain with autophagy markers (ATG8, NBR1) to visualize autophagy-dependent transport

  • Use FM4-64 to track endocytic pathways and confirm vacuolar targeting

• Subcellular fractionation with immunoblotting:

  • Separate cellular components (cytosol, microsomes, PM, vacuole)

  • Quantify EXO70B2 levels in each fraction before and after immune stimulation

  • This has revealed significant redistributions of EXO70B2 after BTH treatment

• Inhibitor studies:

  • Use Concanamycin A to block vacuolar degradation and visualize vacuolar accumulation

  • Apply Brefeldin A (BFA) to study transit through BFA-sensitive compartments

  • These approaches have revealed that EXO70B2 is targeted to the vacuole during immune responses

How can EXO70B2 antibodies be used to study its interaction with the autophagy machinery?

EXO70B2 interacts with autophagy machinery through specific mechanisms:

• Co-immunoprecipitation studies:

  • Use anti-EXO70B2 antibodies to pull down complexes and probe for ATG8

  • Alternatively, perform the reverse IP with anti-ATG8 antibodies

  • These experiments have confirmed that EXO70B2 interacts with ATG8 via ATG8-interacting motifs (AIMs)

• Co-localization analysis:

  • Perform double immunostaining for EXO70B2 and autophagy markers

  • Calculate Pearson's correlation coefficients to quantify co-localization

  • Studies have reported coefficients of 0.48 for ATG8 and 0.4 for NBR1 after BTH/ConcA treatment

• Mutational analysis:

  • Compare localization patterns of wild-type EXO70B2 versus AIM-mutated variants

  • Assess how phosphorylation affects the interaction with autophagy machinery

  • Evidence suggests that phosphorylation increases the interaction with ATG8

• Ultrastructural studies:

  • Use immunogold labeling with anti-EXO70B2 antibodies for electron microscopy

  • This approach has confirmed EXO70B2 transport into the vacuole upon autophagy induction

What are common data inconsistencies when working with EXO70B2 antibodies and how should they be resolved?

Researchers may encounter several types of data inconsistencies:

• Discrepancies between total protein and fractionated samples:

  • Studies have found that while total EXO70B2 levels may not change dramatically after BTH treatment, microsomal fractions show significant increases

  • Resolution: Always analyze both total and fractionated samples to capture redistribution effects

• Variability in subcellular localization:

  • EXO70B2 shows complex localization patterns that change with cellular context

  • In root hairs, localization focuses at the growing tip in distinct foci

  • Resolution: Examine multiple cell types and developmental stages to understand context-specific localization

• Antibody sensitivity limitations:

  • Some pools of EXO70B2 may be below detection limits in certain conditions

  • For example, microsomal EXO70B2 was undetectable in control samples but became detectable after BTH treatment

  • Resolution: Use more sensitive detection methods or concentration steps for low-abundance fractions

• Treatment-dependent effects:

  • Different treatments (BTH, flg22, ConcA) have distinct effects on EXO70B2 dynamics

  • Resolution: Include appropriate controls for each treatment and avoid generalizing results across different immune elicitors

How can I design experiments to distinguish between EXO70B2 and other EXO70 family members?

Distinguishing between different EXO70 proteins requires careful experimental design:

• Antibody selection strategies:

  • Target least conserved regions (typically N-terminal) for maximum specificity

  • Validate against recombinant proteins of multiple family members

  • Consider using epitope-tagged versions when antibody specificity is challenging

• Genetic approaches:

  • Use knockout/knockdown lines for validation (e.g., exo70B2-3)

  • Consider multiple mutants when functional redundancy exists (e.g., EXO70B1 and EXO70B2)

  • Complement with specific variants to confirm antibody specificity

• Comparative analysis:

  • Study multiple EXO70 isoforms in parallel (e.g., EXO70A1, EXO70B1, EXO70B2)

  • This approach has revealed both shared and distinct behaviors during immune responses

  • For example, BTH treatment affects multiple exocyst subunits, not just EXO70B2

• Mass spectrometry validation:

  • When specificity is critical, validate antibody pulldowns with mass spectrometry

  • This can confirm the precise identity of the detected protein and any co-purifying partners

How should researchers approach studying the role of EXO70B2 phosphorylation in different physiological contexts?

Studying context-dependent phosphorylation requires:

• Phosphorylation site mapping:

  • Identify all potential phosphorylation sites using phosphoproteomic approaches

  • Focus on sites that are modified in response to specific stimuli (e.g., flg22)

  • Pay particular attention to MPK3 target sites, which have been linked to immune function

• Conditional expression systems:

  • Generate phospho-mimetic (S/T to D/E) and phospho-null (S/T to A) variants

  • Express these under native or inducible promoters

  • Compare phenotypes in different contexts (normal growth, immune challenge)

• Protein-protein interaction studies:

  • Assess how phosphorylation affects interactions with partners (exocyst components, ATG8, etc.)

  • This approach has revealed that phosphorylation affects both secretion site localization and autophagy targeting

• Phenotypic analysis:

  • Compare resistance to pathogens between wild-type and phospho-variant lines

  • Assess sensitivity to immune elicitors like BTH

  • Studies have shown that phospho-null variants display higher effector-triggered immunity and increased BTH sensitivity

What approaches can be used to study temporal dynamics of EXO70B2 during pathogen infection?

Understanding temporal dynamics requires:

• Time-course experiments:

  • Collect samples at multiple timepoints after pathogen challenge

  • Include both early (minutes to hours) and late (hours to days) timepoints

  • Compare with parallel time courses using purified elicitors (flg22)

• Pulse-chase approaches:

  • Track newly synthesized versus existing EXO70B2 populations

  • This can reveal turnover rates during different phases of the immune response

• Live-cell imaging complementation:

  • While antibodies are not suitable for live imaging, complementary approaches with fluorescent protein fusions can provide dynamic information

  • Compare these patterns with fixed-cell antibody staining at equivalent timepoints

• Quantitative analysis:

  • Measure relative abundance in different subcellular fractions over time

  • Calculate colocalization coefficients with various markers at each timepoint

  • Create mathematical models of EXO70B2 trafficking during immune responses